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Abortive Initiation

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Konstantin Severinov – One of the best experts on this subject based on the ideXlab platform.

  • Role of the RNA polymerase sigma subunit in transcription Initiation
    Research in microbiology, 2002
    Co-Authors: Sergei Borukhov, Konstantin Severinov
    Abstract:

    In bacteria, σ subunits direct the catalytically competent RNA polymerase core enzyme to promoters. Recent advances in our understanding of bacterial RNA polymerase reveal that σ subunits are intimately involved in all aspects of transcription Initiation including promoter location, promoter melting, Initiation of RNA synthesis, Abortive Initiation and promoter escape.

  • T7 RNA polymerase transcription complex: What you see is not what you get
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Konstantin Severinov
    Abstract:

    After promoter-complex formation, all RNAPs undergo Abortive Initiation–a catalytic synthesis of short, 2- to 8-nt-long, RNA oligomers that are rapidly synthesized and released from the complex. When the nascent RNA chain reaches a critical length of about 9 bases, it becomes stably associated with the transcription complex. RNAP then clears the promoter and elongates the nascent RNA chain in a fully processive manner. Despite its tight grip on nucleic acids, the elongating RNAP moves rapidly along the DNA and RNA chains until it encounters a termination signal, which typically consists of an RNA hairpin followed by a run of uridines. At such sites, RNAP transiently pauses, the stability of the elongation complex suddenly decreases, and the enzyme releases nucleic acids. The enzyme is now available to initiate transcription from promoters again.

  • Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA.
    Journal of molecular biology, 1998
    Co-Authors: Elena Severinova, Konstantin Severinov, Seth A. Darst
    Abstract:

    The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, σ70, with high affinity and inhibits its transcription activity. AsiA binds to σ70 primarily through an interaction with σ70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the −35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and σ70 and thus does not inhibit σ70 activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and Abortive Initiation at two −10/−35 type promoters and two “extended −10” promoters. Our results indicate that the binding of AsiA to σ70 and the interaction of σ70 region 4.2 with the −35 consensus promoter element of −10/−35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and Abortive Initiation from extended −10 promoters, which lack a −35 consensus element and do not require σ70 conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP σ70 holoenzyme transcription at −10/−35 promoters by interfering with the required interaction between σ70 conserved region 4.2 and the −35 consensus promoter element.

Seth A. Darst – One of the best experts on this subject based on the ideXlab platform.

  • Structure of a bacterial RNA polymerase holoenzyme open promoter complex
    eLife, 2015
    Co-Authors: Brian Bae, Andrey Feklistov, Agnieszka Lass-napiorkowska, Robert Landick, Seth A. Darst
    Abstract:

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 A-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates Abortive Initiation and σ(A) dissociation.

  • Abortive Initiation using crosslinking nucleotide analog substrates.
    , 2015
    Co-Authors: Matthew J. Bick, Sohail Malik, Arkady Mustaev, Seth A. Darst
    Abstract:

    A. Structure of the photoactivatable crosslinking nucleotide analog substrates used in the present study. B. Abortive Initiation reactions using a minimal transcription system containing the general transcription factors TFIIB, TFIIE, TFIIF, and TBP and Pol II, and the crosslinking nucleotide analogs as substrates. These reactions were carried out without photo-crosslinking. Left to right: Lane 1—UCA initiating nucleotide. Lane 2—UCA crosslinking analog. Lane 3—CA crosslinking analog. Lane 4—no protein control.

  • A minimal transcription system sufficient for Initiation at the Ad ML promoter.
    , 2015
    Co-Authors: Matthew J. Bick, Sohail Malik, Arkady Mustaev, Seth A. Darst
    Abstract:

    A. SDS-PAGE analyses of Pol II and GTFs used in the study. TFIIE subunits (α and β) were expressed and added to reactions separately. TFIIF was reconstituted prior to use. The Pol II preparation was stained with silver; all others with Coomassie. B. All transcription reactions contained Pol II, TBP, TFIIB, TFIIE, and TFIIF; TFIIH and other additions were as indicated. Following standard transcription reactions with templates with the indicated topologies (supercoiled or linearized), full-length (FL) transcripts were processed for electrophoresis on 4% PAGE containing urea. C. Abortive Initiation reactions were primed with the CpA dinucleotide and reactions also contained α32P-CTP. TBP and TFIIB were omitted from control reactions in lanes 9 and 10. TFIIH was added as indicated; other additions were also as indicated. α-ama, α-amanitin.

Lilian M. Hsu – One of the best experts on this subject based on the ideXlab platform.

  • Monitoring Abortive Initiation.
    Methods (San Diego Calif.), 2008
    Co-Authors: Lilian M. Hsu
    Abstract:

    Abstract Abortive Initiation, when first discovered, was an enigmatic phenomenon, but fully three decades hence, it has been shown to be an integral step in the transcript Initiation process intimately tied to the promoter escape reaction undergone by RNA polymerase at the Initiationelongation transition. A detailed understanding of Abortive Initiation-promoter escape has brought within reach a full description of the transcription Initiation mechanism. This enormous progress was the result of convergent biochemical, genetic, and biophysical investigations propelled by parallel advances in quantitation technology. This chapter discusses the knowledge gained through the biochemical approach and a high resolution method that yields quantitative and qualitative information regarding Abortive Initiation-promoter escape at a promoter.

  • In vitro studies of transcript Initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on Abortive Initiation and promoter escape.
    Biochemistry, 2003
    Co-Authors: Lilian M. Hsu, Caroline M. Kane, Michael J. Chamberlin
    Abstract:

    Abortive Initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript Initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative Initiation parameters that describe Abortive Initiation and promoter escape; these parameters are the Abortive rate, the productive rate, the Abortive:productive ratio, the Abortive probability, and the maximum size of Abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the Abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the Abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher Abortive yield, a higher Abortive probability, a longer Abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of Abortive Initiation.

  • In vitro studies of transcript Initiation by Escherichia coli RNA polymerase. 2. Formation and characterization of two distinct classes of initial transcribing complexes.
    Biochemistry, 2003
    Co-Authors: Lilian M. Hsu, Caroline M. Kane, Michael J. Chamberlin
    Abstract:

    By following the kinetics of Abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term “productive ITC” and “unproductive ITC”. The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of Abortive Initiation steps. The unproductive ITCs were found to synthesize mostly Abortive transcripts of 2−3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out Abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproducti…

Michael J. Chamberlin – One of the best experts on this subject based on the ideXlab platform.

  • In vitro studies of transcript Initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on Abortive Initiation and promoter escape.
    Biochemistry, 2003
    Co-Authors: Lilian M. Hsu, Caroline M. Kane, Michael J. Chamberlin
    Abstract:

    Abortive Initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript Initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative Initiation parameters that describe Abortive Initiation and promoter escape; these parameters are the Abortive rate, the productive rate, the Abortive:productive ratio, the Abortive probability, and the maximum size of Abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the Abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the Abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher Abortive yield, a higher Abortive probability, a longer Abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of Abortive Initiation.

  • In vitro studies of transcript Initiation by Escherichia coli RNA polymerase. 2. Formation and characterization of two distinct classes of initial transcribing complexes.
    Biochemistry, 2003
    Co-Authors: Lilian M. Hsu, Caroline M. Kane, Michael J. Chamberlin
    Abstract:

    By following the kinetics of Abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term “productive ITC” and “unproductive ITC”. The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of Abortive Initiation steps. The unproductive ITCs were found to synthesize mostly Abortive transcripts of 2−3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out Abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproducti…

  • Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Lilian M. Hsu, Michael J. Chamberlin
    Abstract:

    The process of RNA chain Initiation by RNA polymerases plays a central role in the regulation of transcription. In this complex phase of transcription, short oligomers are synthesized and released from the enzyme-promoter complex in a reaction termed Abortive Initiation. The polymerase undergoes many cycles of Abortive Initiation prior to completion of the Initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of sigma factor, and formation of an elongation complex in which the RNA is stably bound. We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR. The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro. Clearance from T5 N25antiDSR can be stimulated up to 10-fold in vitro by addition of the E. coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters. Using an E. coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25antiDSR promotor in vivo. The stimulation of RNA chain Initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription Initiation reaction.

Roger D. Kornberg – One of the best experts on this subject based on the ideXlab platform.

  • Initiation Complex Structure and Promoter Proofreading
    Science (New York N.Y.), 2011
    Co-Authors: Xin Liu, David A. Bushnell, Daniel-adriano Silva, Xuhui Huang, Roger D. Kornberg
    Abstract:

    The Initiation of transcription by RNA polypolymerase/a> is a multistage process. X-ray crystal structures of transcription complexes containing short RNAs reveal three structural states: one with 2- and 3-nucleotide RNAs, in which only the 3′-end of the RNA is detectable; a second state with 4- and 5-nucleotide RNAs, with an RNA-DNA hybrid in a grossly distorted conformation; and a third state with RNAs of 6 nucleotides and longer, essentially the same as a stable elongating complex. The transition from the first to the second state correlates with a markedly reduced frequency of Abortive Initiation. The transition from the second to the third state correlates with partial “bubble collapse” and promoter escape. Polymerase structure is permissive for Abortive Initiation, thereby setting a lower limit on polymerase-promoter complex lifetime and allowing the dissociation of nonspecific complexes. Abortive Initiation may be viewed as promoter proofreading, and the structural transitions as checkpoints for promoter control.

  • Structural basis of eukaryotic gene transcription
    FEBS letters, 2004
    Co-Authors: Hinrich Boeger, David A. Bushnell, Kenneth D. Westover, Ralph E. Davis, Joachim Griesenbeck, Yahli Lorch, J. Seth Strattan, Roger D. Kornberg
    Abstract:

    An RNA polypolymerase/a> promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polypolymerase/a>, general transcription factors, and Mediator in a ∼3 MDa transcription Initiation complex. X-ray crystallography has revealed the structure of RNA polypolymerase/a>, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X-ray and electron crystallography have led to a picture of the entire transcription Initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, Abortive Initiation, and promoter escape.

  • Structural Basis of Transcription: Separation of RNA from DNA by RNA Polymerase II
    Science (New York N.Y.), 2004
    Co-Authors: Kenneth D. Westover, David A. Bushnell, Roger D. Kornberg
    Abstract:

    The structure of an RNA polypolymerase/a>-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from Abortive Initiation to promoter escape.